Methods of detecting molecules expressing selected epitopes via fluorescent dyes

ABSTRACT

Methods, systems and kits are provided for detecting molecules expressing a selected epitope in a sample through use of an epitope detector containing a single chain Fv for the selected epitope or a constrained epitope specific CDR attached to an oligonucleotide.

INTRODUCTION

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/624,946, filed Jul. 25, 2000 now U.S. Pat. No. 6,743,592.

BACKGROUND OF THE INVENTION

Traditional methodologies for protein detection and quantificationinclude 2-D gel electrophoresis, mass spectrometry and antibody binding.Each methodology has been used to quantify protein levels fromrelatively large amounts of tissue, yet each suffers from a lack ofsensitivity.

Improvement of the ability to monitor proteins, lipids, sugars andmetabolite levels and their modifications is needed for cell biology andmedicine. A variety of technologies have been employed to improve thesensitivity of detecting these molecules. Recent examples of detectionmethods include immuno-PCR, RCA and immuno-aRNA.

Immuno-PCR (U.S. Pat. No. 5,665,539) combines the polymerase chainreaction (PCR) technology with conventional detection methods toincrease the sensitivity to detect protein. However, a major limitationof immuno-PCR lies in the non-linear amplification ability of PCRreaction, which limits this technique as a quantitative detectionmethod. Thus, this method provides no direct correlation between theamount of signal and the amount of protein present.

A relatively isothermal rolling circle DNA amplification technique (RCA;Schwietzer et al., Proc. Natl. Acad.Sci. USA 97, 10113, 2000)) providesan improvement over immuno-PCR as this technique overcomes some of thequantitation problems associated with immuno-PCR.

U.S. Pat. No. 5,922,553 discloses a method for quantifying levels of aselected protein via a technique referred to as immuno-aRNA. In thismethod, a first antibody targeted to a selected protein is immobilizedto a solid support. The support is then contacted with the selectedprotein so that the selected protein is immobilized to the firstantibody. The solid support is then contacted with a RNA promoter-drivencDNA sequence covalently coupled to a second antibody targeted to theselected protein so that the second antibody binds to the bound selectedprotein. The amount of selected protein is determined by quantifyinglevels of the promoter driven cDNA sequence covalently coupled to thebound second antibody via an amplified RNA technique. In a preferredembodiment, a T7 promoter driven cDNA sequence is covalently coupled tothe second antibody.

It has now been found that single chain fragments as well as exocyclicpeptide based complementarity determining region (CDR) subunits can beused in this immuno-aRNA technique. Further, it has been found that PCR,as well as amplified RNA techniques, can be used to quantify thepromoter driven cDNA sequence covalently coupled to the bound singlechain fragment or CDR subunit. The use of smaller antibody binding unitsand fragments coupled with the already existing large single chain orcyclic peptide libraries and the use of robotic assistance renders thismethod widely useful for both medicinal and research purposes.Furthermore, a single third detector species can be coupled withdouble-stranded DNA and bound to either the single chain Fv or the CDRs,rendering detection uniform and simple.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for detectingmolecules expressing a selected epitope in a sample. In this method, anepitope anchor specific for a selected epitope is immobilized to aselected surface. The epitope anchor may comprise a single chain Fvfragment, a CDR, an antibody, or other ligand peptide or chemical orpharmaceutical that interacts with a selected epitope. The surface isthen contacted with a sample suspected of containing molecules whichexpress the selected epitope so that the molecules bind to theimmobilized epitope anchor. An epitope detector comprising a singlechain Fv for the selected epitope or a constrained epitope specific CDRattached to an oligonucleotide, preferably a double-stranded cDNA, isthen used to detect any bound molecules. Alternatively, the method ofthe present invention can be performed without an epitope anchor. Inthis embodiment, the epitope detector is employed to define moleculesbound directly to a surface.

In a preferred embodiment of this method, detection is performed viareadout using a standard fluorimetric device of a fluorescence derivednumerical value. More specifically, in this embodiment, followingamplification of the oligonucleotide of the epitope detector, afluorescent dye is incorporated into the nucleic acid sequence in alinear manner. Upon excitation at a selected wavelength, the dye yieldsa quanta of fluorescence signals directly proportional to the mass ofsample loaded.

With a mixture of epitopes detectors comprising either monoclonalantibodies for selected epitopes, single chain Fvs for selected epitopesor constrained epitope specific CDRs, conjugated to cDNAs of differentlengths, the method of the present invention can also be used to profileproteins in a cell lysate.

Another object of the present invention is to provide kits for thedetection of molecules expressing a selected epitope which comprise anepitope detector comprising a single chain Fv for the selected epitopeor a constrained epitope specific CDR attached to an oligonucleotide,preferably a double-stranded cDNA. In addition, the kits preferablycomprise an RNA polymerase, an amplification buffer and a fluorescentdye for staining of amplified products. Kits of the present inventionmay further comprise an epitope anchor specific for a selected epitope.In one embodiment, the single chain Fv or the constrained epitopespecific CDR is modified to allow for attachment of the oligonucleotide.In another embodiment, the kit comprises a mixture of epitope detectorsfor profiling of proteins in a cell lysate.

Methods and kits of the present invention can be formulated into aconvenient 96- or 384-well plate platform for high-throughput handlingand screening of samples.

Methods and kits of the present invention are particularly useful inanalyzing the interactions of molecules, e.g. protein-proteininteractions, ligand-induced protein-protein interactions andsugar-protein interactions, as well as for monitoring changes in theseinteractions which are induced by chemicals, pharmaceuticals and othermolecular species including, but not limited to, proteins, lipids andsugars.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to improved methods for quantifying levelsof selected molecules and systems and kits for performing these improvedmethods. In one embodiment, the method comprises binding an epitopeanchor specific for a selected epitope of the molecule to a selectedsurface. The epitope anchor may comprise a single chain Fv fragment, aCDR, an antibody, or other ligand peptide or chemical specific for aselected epitope. In a preferred embodiment, the epitope anchor is boundto a designated spot on the surface. For example, the surface maycomprise a chip and the epitope anchor is bound to a defined spot on thechip. In one embodiment, the epitope anchor is deposited onto a surfaceor plate with the aid of a pipettor or similar device which permitsapplication at a single site. The surface with the bound epitope anchoris then contacted with a sample suspected of containing moleculesexpressing the selected epitope so that the molecule binds to theepitope anchor. In another embodiment, the molecule is attached to asurface directly, without the use of an epitope anchor.

Examples of samples which can be assayed via the methods of the presentinvention include, but are not limited to, individual cells andsolutions including biological fluids such as serum.

An epitope detector which can bind to any bound molecule on the surfaceis then used to detect and quantify the amount of bound molecule. Theepitope detector comprises a monoclonal antibody, a single chain Fv forthe selected epitope or a constrained epitope specific CDR which havebeen modified to allow for attachment of oligonucleotides, preferablydouble-stranded DNA at a single site. Alternatively, fragments ofantibodies with the binding activity, scFv or CDR peptides can be usedto replace antibodies in this technology.

Fv fragments for selected epitopes can be produced in cells or onmicroorganisms by use of recombinant DNA technology. For example, Skerraand Pluckthun (Science 1988 240:1038-1044) describe an expression systemfor production of functional Fv fragments in E. coli.

A method for producing Fv fragments in eukaryotic host cells with aeukaryotic expression vector which has an operon having a DNA sequencewhich encodes the variable domain only of an antibody light or heavychain has also been described (J. Mol. Biol. 1988 203:825-828). Chainsof the Fv fragment are secreted and correctly assembled by the host cellsuch that fully functional Fv fragments are produced in the culturesupernatant. In addition, the DNA coding sequence may be altered towardits 5′ end so that the amino terminal end expresses a residue orresidues with a surface suitable for covalent coupling of anoligonucleotide. In addition, the 3′ terminal end may be varied so thatcysteine residues are produced towards the C-terminal end of eachvariable domain permitting the variable domains in the dimer to becomelinked together by disulphide bonding. This may also promote assembly ofthe Fv fragment. Alternatively, the Fv fragment may be stabilized by useof a vector having a first DNA sequence encoding a first variable domainand a second DNA sequence encoding a second variable domain, the firstand second sequences being linked by a third DNA sequence which encodesa joining peptide sequence. In this case, the joining peptide sequenceis sufficiently long and flexible to allow folding of the twopolypeptides into a functional single chain Fv. Preferably, the hostcell is a myeloma cell line which, prior to transformation, does notsecrete whole antibody or light chains. Such cells lines are well knownand widely available (Reichmann et al. J. Mol. Biol. 1988 203:825-828).

It is believed that random phage technology to any hapten or chemicalcompound can also be used to select Fvs. (Harrison et al. United StatesBiochemical Pharma Ltd. (Europe), Watford, United Kingdom).

The CDR technology is well known and has been described in U.S. Pat. No.5,334,702, U.S. Pat. No. 5,663,144, and U.S. Pat. No. 5,919,764. Ingeneral, CDRs comprise a 6 to 15 mer peptide constrained to be cyclicand modified by aromatic residues. An important step in the design ofconformationally constrained peptides for use in the present inventionis the delineation of the residues that are important for activity. Thisis generally accomplished by first synthesizing a set of analogs fromthe bioactive domain of the original antibody or receptor or ligand ofdifferent lengths and establishing the minimal chain lengths for thecomplete and partial activities. Once the minimal chain length has beenestablished, each side chain can be systematically varied to determinethe importance of charge, steric bulk, hydrophobicity, aromaticity, andchirality at each position. After evaluation of the properties of alarge set of analogs, it is possible to identify the functional groupsand conformation features involved in binding. Differentconformationally constrained analogs can then be developed. Variousmeans for constraining peptides have been developed.

One means involves introducing a conformationally constrained aminoacid. Hruby (Life Sci. 1982 31:189-199) describes the synthesis of alarge number of amino acid and dipeptide derivatives with built-inconformational constraints, as well as their incorporation intobiologically active peptides. Prasad et al. (Biopolymers 1995 35:11-20)also describes a method of constraining the conformation of an aminoacid unit by replacing the hydrogen atom at the α-carbon with a methylgroup to produce a dialkylamino acid. U.S. Pat. No. 6,022,523 describesa method that restricts the conformational freedom of amino acids byintroducing a double-bond at the C-α and C-β atoms.

Another means for constraining peptides involves introduction ofcovalent cross-links. Constraining the peptide backbone by introductionof covalent cross-links provides more dramatic effects thanincorporating unusual amino acids. Macrocyclization is oftenaccomplished by forming an amide bond between the peptide N- andC-termini, between a side chain and the N or C terminus, or between twoside chains. A head-to-tail cyclization of side protected peptidessynthesized by Fmoc/t-butyl solid phase procedures on polysterine resinderivatized with 4-hydroxymethyl-3-methoxyphenoxyacetic acid, the firstgeneration dialkoxy-benzyl linkage agent, has been described bySheppard, R. C. (Int. J. Peptide Res. 1982 20:451-454). In addition, theanalogous linkage agent, 4-(4-hydroxymethyl-3-methoxyphenoxy)-butyricacid (HAMA), was recently employed in fragment condensation and solidphase synthesis of peptides with these highly acid sensitive linkers (InPeptides, E. Giralt and D. Andreu eds, ESCOM, Leiden, The Netherlands1991, 131-133). The enkephalin analogs described by Schiller provide anexample of side-chain to backbone covalent cyclization in which covalentattachment of the e-amino group of the D-lys residue to the C terminalbackbone carboxylate group of Leu produces a cyclic 16-membered ringanalog with high potency and significant μ receptor selectivity(Schiller et al. Int. J. Pep. Prot. Res. 1985; 25:171-177). BOP-reagentand carboimide/1-hydroxy-benzotriazole combinations have also beenreported to be useful in the formation of cyclic peptides (Felix, A. M.Int. J. Pep. Prot. Res. 1988 31:231-238). Degrado et al. have alsodeveloped a biologically active cyclized peptide analog of the GPIIb/IIIa complex using m-aminomethylbenzoic acid as the linker (U.S.Pat. No. 6,022,523).

Disulphides can also be formed by oxidation via introduction of cysteineat certain positions. For example, Romani, S. (Int. J. Pep. Prot. Res.1987 29:107-117) demonstrated that non-symmetrical disulphides can bebuilt with the help of the di-tertbutyl aster of azodicarboxylic acid.Ploux, 0. (Int. J. Pep. Prot. Res. 1987 29:162-169) also describes amethod for formation of non-symmetrical disulphides via thioldisplacement of the 3S-3-nitro-2-pyridinesulfenyl group.

In a preferred embodiment, the oligonucleotide is double-stranded andcomprises a T7 promoter driven cDNA sequence so that it can be amplifiedusing T7 RNA polymerase. In this embodiment, double-stranded cDNA issynthesized for use as a template for T7 RNA polymerase transcription.T7 RNA polymerase requires its promoter site to be double-stranded.

In one embodiment, the site on the Fv or CDR to which theoligonucleotides are attached comprises a series of residues which allowthe attachment of linkers consisting of chemicals such as heterodimericcoupling reagents or other linkers. These residues provide a uniformbinding site for the linker attachment. The linkers attach to this siteand also link oligonucleotides to the Fv or CDR. oligonucleotides may beunmodified or modified. For example, the presence of the amplifiedoligonucleotide can be enhanced by incorporating a beacon or fluorescentlabeled oligonucleotide into the mixture allowing for rapid semiquantitative assessment of the epitope expressing molecules (Ton et al.Chemistry 2000 6:1107-1111; Leone et al. Nucleic Acids Res. 199826(9):2150-2155).

In another embodiment, the oligonucleotide of the epitope detector iscoupled to biotin and the monoclonal antibody, single chain Fv orconstrained epitope specific CDR is coupled to streptavidin andattachment of the oligonucleotide to the monoclonal antibody, singlechain Fv or constrained epitope specific CDR occurs via complexing ofthe biotin to the streptavidin.

Bound epitope detectors may be quantified by methods such asamplification by conventional PCR or aRNA techniques. If the detectionmethod used is immuno aRNA, double-stranded cDNA are preferably used inthe epitope detector. In this embodiment, aRNA is transcribed on thesolid support using a polymerase which recognizes a specific promotersuch as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, or φ29polymerase, unlabeled ribonucleotides, and fluorescently labeledribonucleotides. In another embodiment, the amplified products can serveas templates for further amplification with reverse transcriptase orreplicases to increase the sensitivity.

A variety of means are available for detection of amplified products ofthe epitope detector. In one embodiment, the nucleic acid sequence isdetectably labeled such as with a radioactive label or a fluorescentlabel. In a preferred embodiment, the nucleic acid sequence is notlabeled but rather is stained by fluorescent dye. Other methods such asgel electrophoresis, high performance liquid chromatography,hybridization assays, immunohistochemical assays and/or specific bindingprotein assays can also be used for detection.

Use of Fvs and CDR peptides as the epitope detector renders the methodsof the present invention useful in identifying larger polypeptides thancan be identified by the immuno-aRNA technique of U.S. Pat. No.5,922,553, as well as in identifying motifs in supernatants, fluids,extracts of cells or bacteria or any other eukaryotic organism.Accordingly, the method of the present invention has widespreadapplicability in both medicinal and research purposes. Further, themethod of the present invention is more sensitive than currentlyavailable methods and provides quantitative results.

The ability of Fvs and CDR peptides to serve as epitope detectors ofselected molecules in the method of the present invention wasdemonstrated for the p185 receptor. For these experiments, a singlechain Fv (ScFv) construct of 7.16.4 (designated as 7.16.4 ScFv) wasproduced in accordance with the procedure outlined by Peterson & Greene(DNA and Cell Biology 1998 17(12):1031-1040) wherein the Fv region ofthe heavy chain and light chain region was joined by a (gly4-Ser)5linker. Since ScFv7.16.4 contained a poly-histidine tag, it was purifiedover Ni—NTA resin. After purification, the binding of 7.16.4ScFv wasconfirmed by FACS analysis on B104-1-1 cells, in both direct binding andcompetitive binding against the monoclonal antibody 7.16.4.

AHNP, a constrained exocyclic peptide designed from the CDR3.H region ofthe anti-human p185 antibody 4D5 was also used. AHNP binds to p185 andmimics the growth-inhibitory effects of 4D5 (Park et al. NatureBiotechnology 2000 18:194-198).

Both ScFv7.16.4 and AHNP were coupled to a double-strandedoligonucleotide (ds-oligo) to form epitope detectors. Both ds-oligocoupled 7.16.4ScFv and AHNP were able to detect their antigens, ratp185neu from B104-1-1 cells and human p185her2/neu from T6-17 cells,respectively. Further, conjugation with the ds-oligo to form the epitopedetector did not change the binding affinity of the CDR detectionmolecules with their antigens as determined by plasmon resonanceanalysis. Since 7.16.4ScFv and mAb 7.16.4 have comparable bindingaffinity against the p185 receptor, they were used at comparable molarconcentration in this assay. However, ANHP was used at a higherconcentration since its affinity is lower against p185Her2/neu than 4D5.

A preferred means for detection in the present invention comprisesstaining with a fluorescent dye. In this embodiment, after RNAamplification with a polymerase such as T7 RNA polymerase, T3 RNApolymerase, SP6 RNA polymerase or φ29 polymerase, a portion of thereaction mixture can be mixed with a fluorescent dye such as RiboGreenreagent (Molecular Probes, Inc) (U.S. Pat. No. 5,436,134), aunsymmetrical cyanine dye that binds to RNA directly in the solution andthen releases fluorescence signals. Examples of other fluorescent dyeswith similar properties useful in this method include, but are notlimited to, PicoGreen, TOTO-1 or YOYO-1. The reactions are preferablyperformed in microplates and the fluorescence is read using afluorimeter such as the Spectra Fluora 5 to 15 minutes after mixing RNAsolutions with RiboGreen dye.

In the method of the present invention, a mixture of epitope detectorscomprising either monoclonal antibodies to selected epitopes, singlechain Fvs for selected epitopes, or constrained epitope specific CDRs,conjugated with cDNAs of different lengths can be used in a singlereaction to probe the cell lysate to provide a profile of proteins inthe cell lysate via automatic sequencing. In this method, afteramplification and staining of the amplified products with a fluorescentdye, the reaction mixture is separated by electrophoresis and the sizeof the RNA products are visualized by fluorescent dyes or probes.Moreover, specific probes can hybridize with the reaction mixture andreveal the presence and abundance of the corresponding antigens.

The sensitivity of the fluorescence detection method of the presentinvention was demonstrated for the p185 receptor. In these experiments,a chimeric p185-Fc polypeptide was first coated directly to a 96-wellplate. The 4D5 monoclonal antibody conjugated with a 1 kb cDNA was thenused to detected the chimeric p185-Fc receptor. Since the cDNA containeda T7 promoter, it was amplified by the T7 RNA polymerase. Afteramplification, the RNA product was mixed with the RiboGreen reagent in a96-well plate and excited at 485 nm after 5 to 10 minutes. Thefluorescent reading was collected at 535 nm. Using this method,concentrations as low as 0.177 pg/ml were detected.

Coupling of a ds-oligo directly to the CDR or single chain Fv can be atime-consuming procedure, particularly if the purpose is to detecthundreds or thousands of antigens in a mass screening proteomic assay.In addition, variation in the coupling efficiency can complicate theinterpretation of the amplification results. Accordingly, in a preferredembodiment of the present invention, the Fv or CDR contains a general oruniversal epitope such as an hemagglutinin HA tag or polyhistidine tag.An example of a general or universal epitope is the poly-His-tag in the7.16.4ScFv initially designed for the purification of the protein. Asingle monoclonal antibody or single chain Fv coupled with ds-DNA isthen used to bind to the general epitope to create a universal epitopedetector. The efficacy of a universal epitope detector in the method ofthe present invention was demonstrated with the poly-His-tag in the7.16.4ScFv. In these experiments, p185 receptors were captured by Allcoated on the plate, free 7.16.4ScFv was added, followed by extensivewashing and then incubating with ds-oligo conjugated anti-His antibody.After T7 polymerase amplification, specific bands from lysates of 10⁻⁶dilution of the cells were detected. Accordingly, this sensitivity waseven higher than that seen with the basic protocol without a universalepitope detector.

The method of the present invention is also useful in the detection ofpost-translation modifications. PCR and aRNA techniques were originallydeveloped to detect the activity of target genes at the DNA level. Thesemethods have been adopted exclusively in the application of genomicsresearch, sometimes combined with hybridization. Regardless ofsensitivity, these methods are not able to detect the post-translationmodification at the protein level. Monitoring of such events, however,is very critical since many modifications including, but not limited to,phosphorylation and glycosylation are related to the functional statusof the protein. Thus, experiments were performed to demonstrate theability of the method of the present invention to detect thephosphorylation of the p185 receptor induced by EGF treatment. Asignaling model was established in which, upon EGF stimulation, EGFRheterodimerizes with and trans-activates p185, resulting in thephosphorylation of tyrosine residues on the p185 receptor (Qian et al.Proc. Natl Acad. Sci. 1994 15:1500-1504). The A431 cell line, whichoverexpresses EGFR as well as p185 erbB2, was used in these experiments.After EGF stimulation, the p185 receptor in the cell lysate was capturedby 1E1, a monoclonal antibody developed against p185erB2/neu. PY99, anIgG2b type of anti-phosphorylated Tyr antibody, was used to detectphosphorylated receptors. A second antibody, anti-IgG2b, coupled withds-oligo, was used to probe the antigen-antibody sandwich complex. A431cells stimulated with EGF produced a positive band, which was notobserved in cells without EGF treatment. T6-17 cells, however, alsoshowed a positive band, indicating constitutive phospho-tyrosine on thep185 receptor. These data indicate that this method is capable ofdetecting the functional status of a protein by analyzing itsmodification. Epitope detectors comprising an Fv or CDR coupled to theds-oligo can also be used to detect the functional status of theprotein.

Thus, the present invention provides a sensitive detection method whicheliminates concerns about the non-quantitative nature of immuno-PCRtechniques and which offers vast potential in the field of proteomics.By using a polymerase which recognizes a specific promoter such as T7RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase as well as thespecific promoter in the amplification step, assays performed inaccordance with this method possess linear amplification and precisequantification which are relevant to biological and medical assays. Thenumber of factors that affect the sensitivity of detection have alsobeen reduced. The specific binding between antigens and their Fv ormonovalent CDR is the only critical parameter of this method.

The ability to provide universal epitope detectors provides the methodof the present invention with multiple additional advantages. First, anycellular antigens can be detected without having been first coupled to amonoclonal antibody with ds-oligo. Without the universal probe, themethod would only be useful in looking at one or several particularantigens at a time. The universal probe, on the other hand, allows forthe detection of any cellular or fluid residing antigen with availableFvs or CDRS. In addition, with slight modification in the protocol,different proteins can be detected simultaneously in a singleelectrophoresis lane when oligonucleotides of different sizes areattached to the Fv or CDR of the epitope detector. Thus, as demonstratedherein, the method of the present invention provides a versatiletechnique that is applicable in the identification of protein antigensas well as post-translational modification of polypeptides and otherstructures such as sugars or lipids at the single cell level ofdetection.

The method of the present invention is also useful in the analysis ofinteractions of molecules and the detection of small molecules. Forexample, ligand peptides can be used as epitope detectors on tissuesamples to identify the expression of specific receptors, or verse visa.With available Fvs, CDRS, or binding proteins, small molecules such astoxins or drug metabolites, can be detected in any solution including,but not limited to, water, foods, and body fluids.

To study the interactions of molecules, a two-component (moleculeA/molecule B) interaction system is developed. The two components,molecule A and molecule B may comprise proteins, sugars, or other typesof chemical entities including, but not limited to, carbohydrates, DNAor RNA, or peptides with structural conformations such as alpha helicesor beta-sheets. To develop this two-component system, an epitopedetector such as an antibody for a first molecule, referred to hereafteras molecule A, is placed in proximity with a sample comprising moleculeA so that molecule A binds to the epitope detector. A solutioncontaining products of an expression library constructed so that eachexpressed protein also contains a HA tag or similar tag can then beadded to identify molecules which interact with molecule A. For purposesof the present invention, these molecules are referred to herein asmolecule B or a second molecule. Alternatively, normal cellular extractsor lysates or any fluid containing potential molecule B can be used.

If the second molecule, molecule B, in the library product or cellularextract or lysate binds to the first molecule A bound by the epitopedetector, the new molecules can be detected with either a universaldetector that binds to the tag or marker or by the use of a CDR libraryor a scFv library specific against molecule B. In a preferredembodiment, monitoring of the interaction between molecule A andmolecule B is performed by the fluorescence based quantifiable assaywhich detects the amplified nucleic acid sequence from theoligonucleotide conjugated to the universal detector.

When one of the molecules of the two-component (molecule A/molecule B)interaction system binds a ligand or pharmaceutical drug, thetwo-component system can be used to investigate the effects of theligand or drug on the binding of molecule A to molecule B. Accordingly,the present invention also provides an in vitro system for monitoringdrug affects on interactions of molecules. The ligand or drug can beadded at any step in the assay to determine how the ligand orpharmaceutical drug alters the binding of molecule A to molecule B. Inaddition, more than one drug can be added to the two-componentinteraction system. For example, a second drug such as an antagonist ofthe first drug can be added and the level of binding of A to B as wellas to the complex of A and B can be determined. Further, instead ofknown ligands or drugs, a third solution containing the products ofanother library of molecule C marked with other tags could be added tothe complex of A and B and the effects of this third solution on bindingcan be determined.

Thus, the present invention provides a rapid in vitro screening assaywith a biological readout, namely the formation of a complexinteraction. Further, using this type of system it is possible to buildscreening systems that act like organic analogue computers whose outputis dependent on the number of events developed with each progressiveaddition. These progressive events are disturbed upon addition of athird molecule such as a pharmaceutical drug or ligand that interfereswith this assembly. The quantification of signals before and afteraddition of the third molecule defines the change in output. A positivechange means that the pharmaceutical drug or ligand facilitates thebinding of molecule A to molecule B, while a negative change means thatthe pharmaceutical drug or ligand inhibits the binding of molecule A tomolecule B.

Also provided in the present invention are kits for performing themethods of the present invention. In one embodiment, a kit is providedfor the detection of molecules expressing a selected epitope. In apreferred embodiment, detection of the molecule is performed via afluorescent dye which stains nucleic acid sequences. Thus, this kitpreferably comprises an epitope detector comprising an oligonucleotideattached to a monoclonal antibody for the selected epitope, a singlechain Fv for the epitope or a constrained epitope specific CDR as wellas an RNA polymerase, an amplification reaction buffer, and afluorescent dye. In another embodiment, kits are provided for profilingproteins in a mixture such as a cell lysate. In this embodiment, the kitpreferably comprises a mixture of epitope detectors comprisingmonoclonal antibodies for selected epitopes, single chain Fvs forselected epitopes or constrained epitope specific CDRs conjugated withcDNAs of different lengths, as well as an RNA polymerase, anamplification reaction buffer, and a fluorescent dye.

The original immuno-PCR used pure antigens in the assay. Lateriterations of immuno-PCR examined mixed antigens (Hendrickson et al.Nucleic Acids Research 1999 23(3):522-529) but only showed sensitivityof two to three orders of magnitude higher than ELISA. In a real-worldassay with the background comprising a huge variety of non-specificantigens, sensitivity is always limited by the specificity of the assay.Epitopes bound by the Fvs or CDR fragments are expected to identifylarger polypeptides and can be used to identify motifs in supernatants,fluids, extracts of cells or bacteria or any other eukaryotic organism.Further, actual identity of the polypeptides, organic molecules or sugarstructures can be determined by computer aided analysis of data basesusing the binding of several epitopes by Fvs as a guide. For example,binding by Fv a, d, e, and f would identify a sugar molecule as havingside chains a, d, e, and f, and hence belonging to a family of sugarshaving these same side chains. In this way the present invention allowsdefinition and identification of many, if not all molecules in a cell atany one particular time. Moreover this approach can be used to identifyalternative transcriptional forms translated in an active cell orcellular supernatant. This procedure is easily amenable to 1) use withnonradioactive detection methods, most preferably fluorescent dyes 2)microtized liquid handling procedures, 3) low sample volume detectionsuch as “protein chip” analysis and 4) robotization. For example, a chipcan be developed which contains multiple binding elements or units and asingle universal epitope detector. A binding element that recognizes acommon surface on the monoclonal antibody, the single chain Fv, or theCDR, such as a universal antibody can be used. Alternatively, avidin canby built into each antibody, single chain Fv or CDR and biotin can becoupled to the oligonucleotide. These chips provide the advantage ofmore rapid and higher affinity thereby multiplying the signal.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 Materials

Antibodies used include mAbs 7.16.4 and All, each of which is reactivewith a different epitope in the extracellular domain of p185neu. 1E1 isan IgG1 monoclonal antibody generated against the ectodomain of humanp185her2/neu. rhuMAb 4D5 (Herceptin) was obtained from Genentech. Theanti-phosphotyrosine monoclonal antibody PY99 was obtained from SantaCruz Biotechnology (Santa Cruz, Calif.). The cell line B104-1-1 wasderived from NIH3T3 cell by expressing rat oncogenic p185neu. Theexpression level of p185neu in B104-1-1 cells was determined using an¹²⁵I-labeled anti-neu mAb binding assay. NR6, negative for both EGFR andp185neu, was a subclone from NIH3T3. T6-17 was derived from NIH3T3 byover-expressing the human p185her2/neu receptor. These cell lines wereall cultured in Dulbecco's Modified Eagle's medium (DMEM) containing 10%fetal bovine serum (FBS, Hyclone) at 37° C. in a 5% CO₂ atmosphere.

Example 2 Expression and Purification of His-tagged 7.16.4

LB media (150 ml) containing 50 μg/ml ampicillin was inoculated with a15 ml overnight culture of E. coli DH5 α and maintained at 30° C. untilan optical density of 0.5 (600 nm) was obtained.Isopropyl-β-(-D-thiogalactopyranoside) (IPTG) was added at a finalconcentration of 1 mM to induce His-tagged 7.16.4ScFv. After 3 hours,cells were harvested and lysed by freezing and thawing and resuspendedin 10 ml of urea lysis buffer (10 mM Tris-Cl, pH 8.0, 0.1 M NaH₂PO₄, 1 MNaCl, 8 M urea) supplemented with 0.5 mM phenylmethylsulfonyl fluoride,2 μM pepstatin A, and 2 μM leupeptin. The insoluble cellular debris wasremoved by centrifugation (12,000×g for 15 minutes, followed by 12,000×gfor 30 minutes). The clarified supernatant was mixed with 2 ml of Ni—NTAagarose followed by gentle shaking on ice for 1 hour. The mixture wasloaded into an empty column and unbound protein was washed with urealysis buffer (pH 6.3) at 4° C. His-tagged protein was eluted with urealysis buffer (pH 4.5), and eluate fractions were examined by SDS-PAGEand FACS analysis as described in Example 3. Proteins in the peakfractions were pooled and dialyzed against TKC buffer (50 mM Tris-Cl, pH8.0, 100 mM KCl, 10 mM CaCl₂, 1 mM EDTA, 0.1 mM PMSF).

Example 3 Confirmation of 7.16.4ScFv Binding by FACS Analysis

Cell-surface antigens were detected by Fluorescence-activated cellsorting (FACS). B104-1-1 cells (about 5×10⁵) were incubated (30 minutesat 4° C.) in 200 μl of FACS buffer (PBS containing 0.5% bovine serumalbumin and 0.02% sodium azide) containing the purified 7.16.4ScFv.Cells were then washed in FACS buffer and incubated (30 minutes, 4° C.)with anti-His antibody (Invitrogen). After this second incubation, cellswere washed again in FACS buffer and further incubated with FITC-labeledIgG against mouse immunoglobulins (Sigma Chemical Co., St. Louis, Mo.).After washing with FACS buffer, the cell pellet was suspended in PBSbuffer and processed for analysis by a FACS scan flow cytometer (BectonDickinson). For each sample, 10,000 viable cells were gated followingsize (forward scatter, FSC) and granularity (side scatter, SSC)parameters and analyzed with CellQuest Software (Becton Dickinson). Forcompetitive binding, B104-1-1 cells were first incubated with themonoclonal antibody 7.16.4 in the presence of different concentrationsof 7.16.4ScFv. Cells were then washed in FACS buffer and furtherincubated with FITC-labeled IgG against mouse immunoglobulins (SigmaChemical Co.) before analysis on the flow cytometer.

Example 4 Attachment of ds-cDNA to Antibody or Fv or CDR

An oligonucleotide of the following sequence (GGCTAACTAGAGAACCCACT (SEQID NO:1)) was designed with an activatable amine at the 5′ end to allowfor covalent coupling to primary amines. Another oligonucleotide primer(GCTGGGATCTGTCTCTACAA (SEQ ID NO:2))was derived from an internalsequence from the VCP cDNA. Using these two primers, a cDNA fragment of˜1 kb was amplified from the plasmid pcDNA-VCP. This fragment containsan activatable amine at the 5′ end and a T7 promotor site(TAATACGACTCACTATAGGG (SEQ ID NO:3)) used to direct the synthesis of RNAfrom the cDNA template through the enzymatic activity of T7 RNApolymerase. For attachment of 0.25 μg of antibody to 4 μg of ds cDNA, anequal volume of 0.1% glutaraldehyde was added in 10 μl aliquots. Thesolution was mixed on a rotation device for 3 hours at room temperature.Ethanolamine (1 M; 1/20 volume; pH 7.5) was then added. The solution wasmixed for an additional 2 hours at room temperature. The protein-DNAcomplex was stored at 4° C.

Example 5 Standard Sandwich ELISA as a Control Method

Ninety-six well microtiter plates (Nunc-Immuno Plate, MaxiSorpTM) werecoated with different concentrations of chimeric p185 extra-cellulardomain-Fc polypeptide by incubating plates overnight at 4° C. with 100μl of coating buffer (antibody concentration: 5 μg/ml). Plates were thenwashed three times with PBS containing 0.2% TWEEN 20 (PBS-T; 200μl/well), blocked with PBS containing 0.5% FBS and 0.2% TWEEN 20 (200μl/well) for 1 hour at room temperature, and washed again three timeswith PBS-T (200 μl/well). After this incubation step, plates were washedwith PBS-T (six times, 200 μl/well) and incubated with humanizedanti-p185Her2 antibody 4D5 (150 ng/ml, 50 μl/well) for 2 hours at roomtemperature. Subsequently, plates were washed six times and RNAamplification was performed. The following reagents were added: 1× RNAamplification buffer (50 mM Tris (pH 8.3), 10 mM MgCl₂, 50 mM KCl, 0.5mM Spermidine; 10 mM DTT; 750 AM ATP, UTP, GTP and CTP; 0.5 μl RNAsin;200 U T7 RNA. The solution was then incubated for 4 hours at 37° C. TheRNA product was removed from the wells and 25 μl was diluted with 75 μlTE buffer and then mixed with 100 μl of 2000-fold diluted RiboGreenreagent. Ten minutes later, samples in the 96-well plate were excited at485 nm, and the fluorescence emission was collected at 535 nm using aSpectra Fluora reader (Tecan, Austria).

1. A method for quantifying molecules expressing a selected epitope in asample comprising: (a) immobilizing a molecule expressing a selectedepitope in a sample to a selected surface; (b) contacting the surfacewith an epitope detector so that the epitope detector binds toimmobilized molecules on the surface, said epitope detector comprisingan oligonucleotide attached to a monoclonal antibody for the selectedepitope, a single chain Fv for the epitope or a constrained epitopespecific CDR; (c) amplifying the oligonucleotide of said epitopedetector by RNA amplification; (d) contacting the amplifiedoligonucleotide with a fluorescent dye which binds to RNA and stains theamplified oligonucleotide; and (e) measuring a quanta of fluorescencesignals emitted from the stained oligonucleotide which is directlyproportional to epitope detector bound to the surface and moleculesexpressing the selected epitope in the sample.
 2. A method for detectingmolecules expressing a selected epitope in a sample comprising: (a)immobilizing a molecule expressing a selected epitope in a sample to aselected surface; (b) contacting the surface with an epitope detector sothat the epitope detector binds to immobilized molecules on the surface,said epitope detector comprising an oligonucleotide attached to amonoclonal antibody for the selected epitope, a single chain Fv for theepitope or a constrained epitope specific CDR; (c) amplifying theoligonucleotide of said epitope detector by RNA amplification; (d)adding the amplified oligonucleotide of said epitope detector from step(c) to a reverse transcriptase based reaction or a replicase basedreaction to increase sensitivity; (e) detecting the product of step (d)by containing the product of step (d) with a fluorescent dye or probewhich binds RNA and stains the product of step (d) and measuringfluorescence emitted from the stained product of step (d) which isindicative a epitope detector bound to the surface and moleculesexpressing the selected epitope in the sample.
 3. The method of claim 2wherein the elected surface to which the molecule expressing a selectedepitope in a sample is immobilized is chip or plastic well.
 4. Themethod of claim 1 wherein the selected from the group consisting of: achip and a microtiter plate.
 5. A method for qualifying moleculesexpressing a selected epitope in a sample comprising: (a) immobilizing amolecule expressing a selected epitope in a sample to a selectedsurface; (b) contacting the surface with an epitope detector so that theepitope detector binds to immobilized molecules on the surface, whereinsaid epitope detector is oligonucleotide attached to a single chain Fvthat specifically binds to the selected epitope, or oligonucleotideattached to a constrained epitope specific CDR; (c) amplifying theoligonucleotide of said epitope detector by RNA amplification; (d)contacting the amplified oligonucleotide with a fluorescent dye whichbinds to RNA and stains the amplified oligonucleotide; and (e) measuringfluorescence emitted from the stained oligonucleotide which isindicative of epitope detector bound to the surface and moleculesexpressing the selected epitope in the sample.
 6. The method of claim 5wherein said epitope detector is an oligonucleotide attached to a singlechain Fv that specifically binds to the selected epitope.
 7. The methodof claim 1 wherein the oligonucleotide is linked to the monoclonalantibody, a single chain Fv, or a constrained CDR by biotin-streptavidinlinkers.
 8. The method of claim 1 wherein the oligonucleotide is adouble stranded cDNA molecule.
 9. The method of claim 1 wherein theoligonucleotide comprises an RNA promoter selected from the groupconsisting of: a T7 RNA promoter, a T3 RNA promoter and an SP6 RNApromoter.
 10. The method of claim 1 wherein the fluorescent dye is anunsymmeterical cyanine dye.
 11. A method for detecting moleculesexpressing a selected epitope in a sample comprising: (a) immobilizing amolecule expressing a selected epitope in a sample to a selectedsurface; (b) contacting the surface with an epitope detected so that theepitope detector binds to immobilized molecules on the surface, whereinsaid epitope detector is an oligonucleotide attached to a single chainFv that specifically binds to the selected epitope, or anoligonucleotide attached to a constrained epitope specific CDR; (c)amplifying the oligonucleotide of said epitope detector by RNAamplification; (d) adding the amplified oligonucleotide of said epitopedetector from step (c) to a reverse transcriptase based reaction or areplicase based reaction to increase sensitivity; (e) detecting theproduct of step (d) by contacting the product of step (d) with afluorescent dye or probe which binds RNA and stains the product of step(d) and measuring fluorescence emitted from the stained product of step(d) which is indicative epitope detector bound to the surface andmolecules expressing the selected epitope in the sample.
 12. The methodof claim 11 wherein said epitope detector is an oligonucleotide attachedto a single chain Fv that specifically binds to the selected epitope.13. The method of claim 2 wherein the oligonucleotide is linked to themonoclonal antibody, a single chain Fv, or a constrained CDR bybiotin-streptavidin linkers.
 14. The method of claim 2 wherein theoligonucleotide is a double stranded cDNA molecule.
 15. The method ofclaim 2 wherein the oligonucleotide comprises an RNA promoter selectedfrom the group consisting of: a T7 RNA promoter, a T3 RNA promoter andan SP6 RNA promoter.
 16. The method of claim 2 wherein the fluorescentdye is an unsymmeterical cyanine dye.